Method for producing l-carnitine from crotonobetaine

ABSTRACT

The invention relates to a method for producing L(−)-carnitine. The aim of the invention is to depict a method which permits the synthesis of L(−)carnitine from crotonobetaine. crotonobetaine salts or derivatives in an ecologically advantageous manner by immobilizing cells of  Escherichia coli  044 K74 in a continuously operating cell recycle reactor. Growing or resting cells of  E. coli  are retained in a continuously operating cell recycle reactor by micro or ultrafiltration membranes which are arranged as a flat membrane module or hollow chamfer module.

[0001] The invention relates to a process for producing L-carnitine from crotonobetaine, from salts of crotonobetaine, other derivatives of crotonobetaine or the like.

[0002] It is known that L-carnitine, a ubiquitously occurring compound, plays an important role in metabolism, especially in transporting long-chain fatty acids through the inner mitochondrial membrane. Numerous clinical applications derive from the function of carnitine in the metabolism of eukaryotes, e.g., in the treatment of patients with carnitine deficiency syndromes, in the prevention and therapy of various heart diseases and in the treatment of hemodialysis patients. Further, L-carnitine is significant as a supplemental nutrient and also promotes, as an additive to fermentation media, the growth of yeasts and bacteria. The growing need for this biologically active L-carnitine enantiomer for these and other applications has led to a worldwide search for means of synthesizing this betaine in an optically pure form, since the chemically synthesized racemate cannot be used because it inhibits carnitine acetyl transferase and the carnitine carrier protein.

[0003] To isolate the L-isomer, up to now processes have been used that are based on splitting racemates by fractionated crystallization using optically active acids (e.g., U.S. Pat. No. 4,254,053, 1981), where D(+)-carnitine occurs as a waste product. This problem can be overcome by various biological processes, starting with inexpensive achiral precursors (Adv. Biochem. Eng. Biotechnol., 1993, 50, 21-44). of particular interest is stereospecific hydration of trans-crotonobetaine into L-carnitine using strains of the genera Escherichia (ED 0121444, 1984; DD 221 905, 1987; EP 0320460, 1989) or Proteus (Agric. Biol. Chem., 1988, 52, 2415-2421; U.S. Pat. No. 5,300,430, 1994). The advantage of this method lies in the fact that this achiral precursor can also he obtained by chemical dehydration of the waste product D-carnitine.

[0004] The numerous processes described in the literature with immobilized microorganisms in a continuously operating reactor system have the advantage that

[0005] pure reaction media can he used, thus facilitating the extraction and purification process,

[0006] by using higher concentrations of the biocatalyst in the reaction medium, higher productivities are achieved while the possibility of contamination is reduced,

[0007] there is reduced sensitivity to inhibitors or a nutrient deficiency,

[0008] a higher stability of the biocatalyst is achieved.

[0009] The advantages mentioned can also be applied to a commercially used process.

[0010] A continuously operating reactor in which microorganisms are retained by micro- or ultrafiltration membranes is an immobilization process which, besides the above-mentioned advantage, also entails lower costs for the immobilization while making it possible to have a very slight upscaling.

[0011] Consequently the object of the invention is a process for producing L-carnitine from crotonobetaine, crotonobetaine salts or other crotonobetaine derivatives in a continuous reactor with free or immobilized cells, growing or resting Escherichia coli 044K74 (DSM 8828) cells, that are retained by micro- or ultrafiltration membranes in a flat membrane or hollow fiber module.

[0012]E. coli is kept in the reactor mentioned at temperatures between 20 and 40° C., pH values between pH 6.0-8.0 and under anaerobic conditions that are necessary for the induction of the enzyme that metabolizes carnitine.

[0013] A minimal or complex medium is used as the reaction medium. In both cases, crotonobetaine, crotonobetaine salts or other crotonobetaine derivatives are added in concentrations between 25 mmol and 1M. The minimal medium contains varying concentrations of caseine hydrolyzate and salts (NH₄)₂SO₄, KH₂PO₄, K₂HPO₄, MgSO₄×7H₂O, MnSO₄×4H₂O, FeSO₄×7H₂O, while the complex medium contains varying concentrations of pancreatic peptone and NaCl. To improve the growth of E. coli, glycerine, glucose, ribose, saccharose or lactose are added. Also added to the medium are inhibitors that prevent the transformation of crotonobetaine into γ-butyrobetaine (fumarate, glucose or nitrate) and inductors of carnitine-netabolizing enzymes such as D-, L-, DL-carnitine, their salts and derivatives or crotonobetaine, its salts or derivatives.

[0014] The course of the reaction in the continuous cell-recycle reactor used here can be divided into two stages. The one stage consists of a reactor tank in which cells of E. coli, together with the reaction medium, convert most of the crotonobetaine into L-carnitine. This reactor tank has monitoring elements for pH value, temperature and stirring speed and for the monitoring and correction of oxygen concentration. The feed of the reaction medium into the reactor is performed with a dosing pump- When necessary, excess medium must be removed from the reactor tank. The second stage consists of an external recycling loop that is connected to the reactor tank and conveys the contents of the reactor through a filter unit by means of a pump. While the filtrate is being collected, to isolate L-carnitine from it as the reaction product, the residue from the filtration is fed again to the reactor. For filtering the cell suspension, commercial filter systems of varying provenance can be used as long as they have a pore size below the cell size of E. coli. The speed of the recycling pump remains unchanged to achieve the best possible filtration rates and to minimize the formation of a polarization membrane during the filtration process.

[0015] The expression free E. coli cells indicates the state in which whole cells are suspended in the reaction medium without preventing a cell outflow through the exit solution. The expression immobilized cells describes the state in which whole cells are bonded to soluble polymers or insoluble carriers, or are enclosed in membrane systems (in Methods in Enzymol. 1987, vol. 135, 3-30).

[0016] The concept growth conditions is defined as the situation in which whole cells use substrates and form products during their life cycle. Resting cells are understood as intact cells that are not growing and that show, under certain conditions, special metabolic functions (in “Biotechnology” (Kieslich, K.; Eds. Rehm, H. J. and Reed, G.) Verlag Chemie, Weinheim, Germany. 1984, Vol. 6a, 5-30).

[0017] The process is described below with several embodiments:

EXAMPLE 1

[0018]Escherichia coli 044 K74 is cultivated in an Erlenmeyer flask that is filled to the top and sealed air-tight at 37° C. under anaerobic conditions, on a rotating shaker (150 r.p.m.). The complex medium used has the following composition: 50 mM of crotonobetaine, 50 mM fumarate, 5 g/l of NaCl and varying concentrations (between 0.5 and 10 g/l) of pancreatic peptone. The pH is set using KOH to pH 7.5. Table 1 summarizes the specific growth rates at varying peptone concentrations. TABLE 1 Maximum specific growth rates of Escherichia coli 044 K74 Peptone (g/l) 0.5 1.0 2.5 5.0 10.0 μmax (h⁻¹) 0.224 0.296 0.351 0.372 0.325

[0019] Under the conditions described, growing cells of E. coli are able to produce 20-30 mM of L-carnitine up to the end of the test.

[0020] Concentrations higher than 5 g/l of peptone yield similar growth and kinetic parameters as well as biomass content (OD 600 nm). In contrast, at lower peptone concentrations, lower growth parameters are obtained.

EXAMPLE 2

[0021]Escherichia coli 044 K74 is cultivated in an Erlenmeyer flask that is filled to the top and sealed air-tight at 37° C. under anaerobic conditions, on a rotating shaker (150 r.p.m.). The complex medium used has the following composition: 50 mM of crotonobetaine, 5 g/l of pancreatic peptone, 5 g/l of NaCl and graduated concentrations (between 0 and 75 mM) of fumarate. The pH is set using KOH to pH 7.5.

[0022] Table 2 shows that adding fumarate causes higher growth rates of E. coli 044 K74 and an OD of 600 nm of almost 1.0 in the steady state. Further, fumarate causes L-carnitine formation of 20-30 mM up to the end of the test. In the absence of fumarate, a carnitine concentration of only 5 mM is obtained, TABLE 2 Biomass (OD_(600 nm)) at varying fumarate concentrations after a 10 hour test. Fumarate (mM) 0 25 50 75 μmax (h⁻¹) 0.21 0.37 0.38 0.39 Biomass OD (600 nm) 0.980 0.910 1.00 0.950

EXAMPLE 3

[0023] The ability of Escherichia coli 044 K74 to form L-carnitine from crotonobetaine is induced by crotonobetaine. The induction studies were performed with crotonobetaine between 5 and 75 mM using resting cells. At higher crotonobetaine concentrations, conversion rates of above 60% of L-carnitine are achieved (Table 3). TABLE 3 Production of L-carnitine by resting cells of Escherichia coli 044 K74 as a function of varying crotonobetaine concentrations. Crotonobetaine (mM) 5 25 50 75 L-carnitine 55 60 62 65 production (%)

EXAMPLE 4

[0024]Escherichia coli 044 K74 is cultivated in an Erlenmeyer flask that is filled to the top and sealed air-tight at 37° C. under anaerobic conditions, on a rotating shaker (150 r.p.m.). The complex medium used has the following composition: 50 mM of crotonobetaine, 5 g/l of pancreatic peptone, 5 g/l of NaCl and 50 mM of fumarate. The pH is set using KOH to pH 7.5.

[0025] To raise the biocatalyst concentration in the reactor and to make it possible to have L-carnitine production at dilution rates higher than the maximum specific growth rate, a membrane reactor was used. The cells were retained with a polysulfone microfiltration membrane with an exclusion threshold of 0.1 μm and were used again. The membranes were arranged in a plate module. Table 4 summarizes the biomass content and Table 5 summarizes the carnitine production, the crotonobetaine conversion and the productivity in this system. TABLE 4 Biomass content of E. coli 044 K74 in a continuously operating membrane reactor. Dilution rate (h⁻¹) 0.0 0.2 1.0 2.0 Biomass (g_(dry weight)/l) 0.5 2.1 9.4 27.0

[0026] TABLE 5 L-carnitine production, crotonobetaine conversion and productivity in a continucualy operating cell reactor with Escerichia coli 044 K74 Dilution rate (h⁻¹) 0.0 0.5 1.0 1.5 2.0 L-carnitine 0.0 38 42 42 38 production (%) Crotonobetaine 0.0 24 26 26 30 conversion (%) Productivity 0.0 1.75 3.5 5.5 6.5 (g/l_(reactor)/h)

[0027] It can be seen from the tables that, with immobilized cells of Escherichia coli 044 K74 in a cell recycle reactor, 6.5 l/h of L-carnitine was formed from crotonobetaine with a metabolization rate of almost 40%. 

1. Process for the production of L-carnitine from crotonobetaine, characterized in that an immobilized microorganism converts the starting material crotonobetaine as such, or in the form of its salts or derivatives, into L-carnitine in a continuously operating cell reactor, and ceramics, glass heads, polyurethane disks are used as the supporting material.
 2. Process according to claim 1, wherein the microorganism is E. coli 044 K74 (DSM 8828).
 3. Process according to claim 1 and 2, wherein E. coli is immobilized on supporting material that does not impair its viability.
 4. Process according to claim 1 to 3, wherein the reaction mixture of ceramics that support that microorganism is filtered, the L-carnitine is separated in a way known in the art, and the crotonobetaine is recycled.
 5. Process according to claim 1 to 4, wherein the crotonobetaine concentration in the reaction medium is between 25 mM and 1 M.
 6. Process according to claim 1 to 5, wherein electron acceptors of respiration of inhibitors such as fumarate, nitrate, oxygen, N-oxides or glucose that prevent the hydration of crotonobetaine into γ-butyrobetaine are added to the usual, commercial complex medium or well defined E. coli minimal medium flowing to the reactor,
 7. Process according to claim 1 to 6, wherein inductors of carnitine-metabolizing enzymes such as L-, D-, or DL-carnitine, derivatives and -salts, as well as crotonobetaine, -derivatives, and -salts, are added to the minimal medium.
 8. Process according to claim 1, wherein E. coli 044 K74 is cultivated on a complex medium anaerobically or partially anaerobically, and a fumarate concentration or 50 mM is obtained.
 9. Process according to claim 1, wherein E. coli 044 K74 is cultivated on a complex medium anaerobically or partially anaerobically, and the complex medium has 50 mM of crotonobetaine, 5 g/l of pancreatic peptone, 5 g/l of NaCl and 50 mM of fumarate, a pH of 7.5, and the reaction occurs in a continuously operating membrane reactor.
 10. Process according to claim 1 to 7, wherein the recycling of the bacteria to the reactor is performed continuously using commercial cross current filtration or hollow-fiber modules consisting of ultra- or microfiltration membranes known in the art of varying chemical composition such as cellulose, polysulphone or polysalphonized polysulphone with an exclusion threshold of 300 k1Da or 0.2 ll.
 11. Process according to claim 1 to 9, wherein the synthesis is performed under anaerobic or partially anaerobic conditions.
 12. Process according to claim 1, wherein carnitine production is performed in a continuously operating reactor at varying dilution rates established by two pumps, the dosing and filtering pump, and at varying stirring speeds and biomass concentrations.
 13. Process according to claim 12, wherein the outflow rate of the filtration stream is verified by process control. 